Neutron-scattering studies reveal further details of the Ca2+/calmodulin-dependent activation mechanism of myosin light chain kinase

Joanna K. Krueger, Gang Zhi, James T. Stull, Jill Trewhella

Research output: Contribution to journalArticle

42 Citations (Scopus)

Abstract

Previously, we utilized small-angle X-ray scattering and neutron scattering with contrast variation to obtain the first low-resolution structure of 4Ca2+·calmodulin (CAM) complexed with a functional enzyme, an enzymatically active truncation mutant of skeletal muscle myosin light chain kinase (MLCK). These experiments showed that, upon binding to MLCK, CaM undergoes a conformational collapse identical to that observed when CAM binds to the isolated peptide corresponding to the CaM binding sequence of MLCK. CaM thereby was shown to release the inhibition of the kinase by inducing a significant movement of its CaM binding and autoinhibitory sequences away from the surface of the catalytic core [Krueger, J. K., Olah, G. A., Rokop, S. E., Zhi, G., Stull, J. T., and Trewhella, J. (1997) Biochemistry 36, 6017- 6023]. We report here similar scattering experiments on the CaM·MLCK complex with the addition of substrates; a nonhydrolyzable analogue of adenosine- triphosphate, AMPPNP, and a peptide substrate for MLCK, a phosphorylation sequence from myosin regulatory light chain (pRLC). These substrates are shown to induce an overall compaction of the complex. The separation of the centers-of-mass of the CaM and MLCK components is shortened (by ~12 Å), thus bringing CaM closer to the catalytic site compared to the complex without substrates. In addition, there appears to be a reorientation of CaM with respect to the kinase upon substrate binding that results in interactions between the N-terminal sequence of CaM and the kinase that were not observed in the complex without substrates. Finally, the kinase itself becomes more compact in the CaM·MLCK·pRLC·AMPPNP complex compared to the complex without substrates. This observed compaction of MLCK upon substrate binding is similar to that arising from the closure of the catalytic cleft in cAMP-dependent protein kinase upon binding pseudosubstrate.

Original languageEnglish (US)
Pages (from-to)13997-14004
Number of pages8
JournalBiochemistry
Volume37
Issue number40
DOIs
StatePublished - Oct 6 1998

Fingerprint

Myosin-Light-Chain Kinase
Neutrons
Calmodulin
Neutron scattering
Chemical activation
Phosphotransferases
Substrates
Catalytic Domain
Skeletal Muscle Myosins
Adenylyl Imidodiphosphate
Myosin Light Chains
Peptides
Compaction
Cyclic AMP-Dependent Protein Kinases
Biochemistry
Phosphorylation
Adenosinetriphosphate
Adenosine Triphosphate
X ray scattering
X-Rays

ASJC Scopus subject areas

  • Biochemistry

Cite this

Neutron-scattering studies reveal further details of the Ca2+/calmodulin-dependent activation mechanism of myosin light chain kinase. / Krueger, Joanna K.; Zhi, Gang; Stull, James T.; Trewhella, Jill.

In: Biochemistry, Vol. 37, No. 40, 06.10.1998, p. 13997-14004.

Research output: Contribution to journalArticle

@article{b6de597c91f5415c9bf4f5ee1eb9f3d1,
title = "Neutron-scattering studies reveal further details of the Ca2+/calmodulin-dependent activation mechanism of myosin light chain kinase",
abstract = "Previously, we utilized small-angle X-ray scattering and neutron scattering with contrast variation to obtain the first low-resolution structure of 4Ca2+·calmodulin (CAM) complexed with a functional enzyme, an enzymatically active truncation mutant of skeletal muscle myosin light chain kinase (MLCK). These experiments showed that, upon binding to MLCK, CaM undergoes a conformational collapse identical to that observed when CAM binds to the isolated peptide corresponding to the CaM binding sequence of MLCK. CaM thereby was shown to release the inhibition of the kinase by inducing a significant movement of its CaM binding and autoinhibitory sequences away from the surface of the catalytic core [Krueger, J. K., Olah, G. A., Rokop, S. E., Zhi, G., Stull, J. T., and Trewhella, J. (1997) Biochemistry 36, 6017- 6023]. We report here similar scattering experiments on the CaM·MLCK complex with the addition of substrates; a nonhydrolyzable analogue of adenosine- triphosphate, AMPPNP, and a peptide substrate for MLCK, a phosphorylation sequence from myosin regulatory light chain (pRLC). These substrates are shown to induce an overall compaction of the complex. The separation of the centers-of-mass of the CaM and MLCK components is shortened (by ~12 {\AA}), thus bringing CaM closer to the catalytic site compared to the complex without substrates. In addition, there appears to be a reorientation of CaM with respect to the kinase upon substrate binding that results in interactions between the N-terminal sequence of CaM and the kinase that were not observed in the complex without substrates. Finally, the kinase itself becomes more compact in the CaM·MLCK·pRLC·AMPPNP complex compared to the complex without substrates. This observed compaction of MLCK upon substrate binding is similar to that arising from the closure of the catalytic cleft in cAMP-dependent protein kinase upon binding pseudosubstrate.",
author = "Krueger, {Joanna K.} and Gang Zhi and Stull, {James T.} and Jill Trewhella",
year = "1998",
month = "10",
day = "6",
doi = "10.1021/bi981311d",
language = "English (US)",
volume = "37",
pages = "13997--14004",
journal = "Biochemistry",
issn = "0006-2960",
publisher = "American Chemical Society",
number = "40",

}

TY - JOUR

T1 - Neutron-scattering studies reveal further details of the Ca2+/calmodulin-dependent activation mechanism of myosin light chain kinase

AU - Krueger, Joanna K.

AU - Zhi, Gang

AU - Stull, James T.

AU - Trewhella, Jill

PY - 1998/10/6

Y1 - 1998/10/6

N2 - Previously, we utilized small-angle X-ray scattering and neutron scattering with contrast variation to obtain the first low-resolution structure of 4Ca2+·calmodulin (CAM) complexed with a functional enzyme, an enzymatically active truncation mutant of skeletal muscle myosin light chain kinase (MLCK). These experiments showed that, upon binding to MLCK, CaM undergoes a conformational collapse identical to that observed when CAM binds to the isolated peptide corresponding to the CaM binding sequence of MLCK. CaM thereby was shown to release the inhibition of the kinase by inducing a significant movement of its CaM binding and autoinhibitory sequences away from the surface of the catalytic core [Krueger, J. K., Olah, G. A., Rokop, S. E., Zhi, G., Stull, J. T., and Trewhella, J. (1997) Biochemistry 36, 6017- 6023]. We report here similar scattering experiments on the CaM·MLCK complex with the addition of substrates; a nonhydrolyzable analogue of adenosine- triphosphate, AMPPNP, and a peptide substrate for MLCK, a phosphorylation sequence from myosin regulatory light chain (pRLC). These substrates are shown to induce an overall compaction of the complex. The separation of the centers-of-mass of the CaM and MLCK components is shortened (by ~12 Å), thus bringing CaM closer to the catalytic site compared to the complex without substrates. In addition, there appears to be a reorientation of CaM with respect to the kinase upon substrate binding that results in interactions between the N-terminal sequence of CaM and the kinase that were not observed in the complex without substrates. Finally, the kinase itself becomes more compact in the CaM·MLCK·pRLC·AMPPNP complex compared to the complex without substrates. This observed compaction of MLCK upon substrate binding is similar to that arising from the closure of the catalytic cleft in cAMP-dependent protein kinase upon binding pseudosubstrate.

AB - Previously, we utilized small-angle X-ray scattering and neutron scattering with contrast variation to obtain the first low-resolution structure of 4Ca2+·calmodulin (CAM) complexed with a functional enzyme, an enzymatically active truncation mutant of skeletal muscle myosin light chain kinase (MLCK). These experiments showed that, upon binding to MLCK, CaM undergoes a conformational collapse identical to that observed when CAM binds to the isolated peptide corresponding to the CaM binding sequence of MLCK. CaM thereby was shown to release the inhibition of the kinase by inducing a significant movement of its CaM binding and autoinhibitory sequences away from the surface of the catalytic core [Krueger, J. K., Olah, G. A., Rokop, S. E., Zhi, G., Stull, J. T., and Trewhella, J. (1997) Biochemistry 36, 6017- 6023]. We report here similar scattering experiments on the CaM·MLCK complex with the addition of substrates; a nonhydrolyzable analogue of adenosine- triphosphate, AMPPNP, and a peptide substrate for MLCK, a phosphorylation sequence from myosin regulatory light chain (pRLC). These substrates are shown to induce an overall compaction of the complex. The separation of the centers-of-mass of the CaM and MLCK components is shortened (by ~12 Å), thus bringing CaM closer to the catalytic site compared to the complex without substrates. In addition, there appears to be a reorientation of CaM with respect to the kinase upon substrate binding that results in interactions between the N-terminal sequence of CaM and the kinase that were not observed in the complex without substrates. Finally, the kinase itself becomes more compact in the CaM·MLCK·pRLC·AMPPNP complex compared to the complex without substrates. This observed compaction of MLCK upon substrate binding is similar to that arising from the closure of the catalytic cleft in cAMP-dependent protein kinase upon binding pseudosubstrate.

UR - http://www.scopus.com/inward/record.url?scp=0032491167&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=0032491167&partnerID=8YFLogxK

U2 - 10.1021/bi981311d

DO - 10.1021/bi981311d

M3 - Article

VL - 37

SP - 13997

EP - 14004

JO - Biochemistry

JF - Biochemistry

SN - 0006-2960

IS - 40

ER -